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Discussion

The present study shows that treatment of PC12 cells with FA leads to (i) cytotoxicity; (ii)
inhibition of CBS expression and decrease in endogenous H2S production; and (iii) increase in the
levels of nNOS and iNOS and overproduction of NO. Knockdown of CBS using shRNA
deteriorates FA-induced decreases in endogenous H2S generation, neurotoxicity, and intracellular
ROS accumulation in PC12 cells. We also show that inhibition of NOS by its specific inhibitor
(ADMA) significantly attenuates FA-induced decreases in endogenous H2S generation,
neurotoxicity, and intracellular ROS accumulation in PC12 cells. Collectively, these findings
suggest that FA confers neurotoxicity by inhibiting the generation of H2S through overproduction
of NO.

Our previous findings that treatment of PC12 cells with FA reduces the viability of cell and
promotes the release of LDH demonstrate the neurotoxicity of FA. It has been shown that FA
negatively affects learning and memory in animals [10], [42] and siginficantly leads to neuronal
damage in the prefrontal cortex of rats including the hippocampus [8], [9]. Song and colleagues
reported that exposure of the cultured cortical neurons to FA causes neurotoxic effects [4]. These
previous results provide strong evidence to support the neurotoxicity of FA. However, the
mechanisms involved in FA toxicity to neural cells have not been fully clarified.

H2S, characterized with an odor of rotten eggs, is a poisonous gas used as chemical reagent.
Nevertheless, more and more literatures reported that H2S involves in many physiological
functions, especially in the nervous system and cardiovascular system [43], [44]. H2S has been
referred to as the third gaseous signaling molecule alongside NO and carbon monoxide
(CO) [43], [45], [46]. In the present study, we showed that FA decreases the generation of H2S in
PC12 cells. CBS is the major enzyme responsible for endogenous H2S generation in PC12
cells [31]. In this study, we also demonstrated the downregulation of CBS expression in FA-treated
PC12 cells. These data indicated that FA disturbs the generation of endogenous H2S in PC12 cells
by inhibition of CBS expression. It has been reported that oxidative damage is one of the most
critical effects of FA exposure [18]. Lu and colleagues have demonstrated that FA causes deficit
in learning and memory by oxidative stress [10]. Our previous results have demonstrated that PC12
cells exposed to FA has a significant increase in intracellular ROS accumulation and indicated that
FA-caused oxidative stress plays a critical role in its neurotoxicity [5]. Oxidative stress occurs
from either a overproduction of ROS or a decline in antioxidant defenses [47]. Unbalanced in
proportion to protective antioxidants causes the excess ROS and ultimately leads to cell
death [20], [21]. H2S is an endogenous antioxidant gas [22]. Thus, we hypothesize that FA-
inhibited H2S generation is involved in its neurotoxicity.

To confirm whether the disturbance of H2S synthesis is a novel mechanism underlying FA-induced
neurotoxicity, we investigated the effects of inhibited H2S generation by CBS knockdown on the
neurotoxicity of FA. In this present work, we showed that inhibition of endogenous H2S production
by CBS silencing using CBS siRNA aggravates FA-induced cytotoxicity, apoptosis, and
accumulation of intracellular ROS in PC12 cells. Our previous results that treatment with NaHS,
a H2S donor, suppresses FA-induced cytotoxicity, apoptosis, loss of mitochondrial membrane
potential, and accumulation of intracellular ROS have demonstrated that exogenous application of
H2S reverses the neurotoxicity and oxidative stress induced by FA [48]. Taken together, our
previous and present findings indicate that FA-disturbed H2S production plays an important role
in its neurotoxicity. Disturbed H2S synthesis has been involved in Downs syndrome [49],
stroke [50] and possibly Alzheimers disease [51][53]. We have previously demonstrated that
inhibition of H2S production contributes to the neurotoxicity induced by MPP+ and
homocystene [31], [32]. Those previous findings provide reasonable explanation for our results.
Thus, we suggest that FA-induced inhibition of CBS expression and decrease in endogenous H2S
generation results in deficiency in ROS scavenging, which in turn leads to ROS accumulation and
ultimately causes neurotoxic effect.

In the present study, we further examined the mechanism of FA-induced disturbance of H2S
synthesis. It has been reported that NO is able to inhibit CBS activity [37], [38]. Recently, the
result from Songur demonstrated that inhalation of FA significantly increases the level of NO in
the cerebellar tissue [36]. It is logical for us to assume that overproduction of NO is responsible
for the role of FA in the disturbance of H2S synthesis in PC12 cells. In the present work, we showed
that treatment of PC12 with FA leads to excess of NO production, which is consistent with the
result from Songur [36]. Since the expression of CBS and the activity of H2S generation in PC12
cells were significantly suppressed by treatment with FA, we hypothesized that the elevated NO
level during FA treatment might play roles in downregulating the expression of CBS leading to
decrease in H2S generation. Levels of NO remained elevated in PC12 cells during FA treatment
periods, while a significant increase in the levels of nNOS and iNOS was also detected in the FA-
exposed PC12 cells. To confirm whether the elevation of NO production mediates the decrease of
H2S synthesis, we investigated the effects of ADMA, a specifical NOS inhibitor, on FA-induced
elevation of nNOS and iNOS levels and NO generation as well as inhibition of endogenous H2S
production in PC12 cells. Our present data showed that pretreatment with ADMA not only reverses
FA-induced increase in the levels of nNOS and iNOS and the production of NO but also recovers
the expression of CBS and the endogenous generation of H2S. Therefore, these findings revealed
that FA-induced NO overproduction contributes to its roles in inhibiting the expression of CBS
and the generation of H2S in PC12 cells. NO and H2S are a growing family of regulatory gaseous
molecules involved in regulation of physiological and pathological functions in mammalian
tissues. They can not only exert comparable biological actions but also compete with and are
antagonists with each other [54]. Recently, it has been demonstrated that H2S directly inhibits the
generation of NO [55]. Our data provide a novel aspect for understanding of the interaction
between two gasotransmitters, H2S and NO.

Finally, we explored whether inhibition of FA-induced NO overproduction by ADMA could


ameliorate the neurotoxicity and oxidative stress induced by FA. The present results demonstrated
that pretreatment with ADMA to inhibit FA-elicited NO overproduction prevents FA-caused
cytotoxicity, apoptosis, and accumulation of ROS in PC12 cells. These findings further support
the notion that NO, at elevated levels, contributes to inhibition of CBS activity and decrease in
H2S generation, which in turn results in ROS accumulation and ultimately leads to neurotoxic
effect.

In conclusion, the present work showed that FA inhibits the expression of CBS and the generation
of H2S as well as elevates the production of NO. Inhibition of H2S generation by knockdown of
CBS aggravated the neurotoxicity of FA, while suppressed NO overproduction by ADMA, a
specifical NOS inhibitor, not only rescued FA-induced downregulation of CBS and decrease in
H2S generation but also prevented the neurotoxicity of FA. These results clearly identify that the
neurotoxicity of FA is contributed to the inhibited endogenous H2S production, which is mediated
by overproduction of NO. The present findings offer a novel mechanistic explanation to the
neurotoxicity of FA. Based on the current findings, we propose that novel therapeutic approaches
for FA neurotoxicity should focus on the restoration of NO and H2S homeostasis in neurons.